The invention relates to antigens, means and methods for the diagnosis of lymphadenopathy and acquired immune deficiency syndrome.
The acquired immune deficiency syndrome (AIDS) has recently been recognized in several countries. The disease has been reported mainly in homosexual males with multiple partners, and epidemiological studies suggest horizontal transmission by sexual routes as well as by intravenous drug administration, and blood transfusion. The pronounced depression of cellular immunity that occurs in patients with AIDS and the quantitative modifications of subpopulations of their T lymphocytes suggest that T cells or a subset of T cells might be a preferential target for the putative infectious agent. Alternatively, these modifications may result from subsequent infections. The depressed cellular immunity may result in serious opportunistic infections in AIDS patients, many of whom develop Kaposi""s sarcoma. However, a picture of persistent multiple lymphadenopathies has also been described in homosexual males and infants who may or may not develop AIDS. The histological aspect of such lymph nodes is that of reactive hyperplasia. Such cases may correspond to an early or a milder form of the disease.
It has been found that one of the major etiological agents of AIDS and of lymphadenopathy syndrom (LAS), which is often considered as a prodromic sign of AIDS, should consist of a T-lymphotropic retrovirus which has been isolated from a lymph node of a homosexual patient with multiple lymphadenopathies. The virus appears to be distinct from the human T-cell leukemia virus (HTLV) family (R. C. Gallo and M. S. Reitz, xe2x80x9cJ. Natl. Cancer Inst.xe2x80x9d, 69 (No. 6), 1209 (1982)). The last mentioned virus has been known as belonging to the so-called HTLV-1 subgroup.
The patient was a 33-year-old homosexual male who sought medical consultation in December 1982 for cervical lymphadenopathy and asthenia (patient 1). Examination showed axillary and inguinal lymphadenopathies. Neither fever nor recent loss of weight were noted. The patient had a history of several episodes of gonorrhea and had been treated for syphilis in September 1982. During interviews he indicated that he had had more than 50 sexual partners per year and had travelled to many countries, including North Africa, Greece, and India. His last trip to New York was in 1979.
Laboratory tests indicated positive serology (immunoglobulin G) for cytomegalovirus (CMV) and Epstein-Barr virus. Herpes simplex virus was detected in cells from his throat that were cultured on human and monkey cells. A biopsy of a cervical lymph node was performed. One sample served for histological examination, which revealed follicular hyperplasia without change of the general structure of the lymph node. Immunohistological studies revealed, in paracortical areas, numerous T lymphocytes (OKT3+). Typing of the whole cellular suspension indicated that 62 percent of the cells were T lymphocytes (OKT3+), 44 percent were T-helper cells (OKT4+), and 16 percent were suppressor cells (OKT8+).
Cells of the same biopsed lymph node were put in culture medium with phytohemagglutinin (PHA), T-cell growth factor. (TCGF), and antiserum to human xcex1 interferon (xe2x80x9cThe cells were grown in RPMI-1640 medium supplemented with antibiotics, 10xe2x88x925M xcex2-mercaptoethanol, 10 percent fetal calf serum, 0.1 percent sheep antibody to human xcex1 interferon (neutralizing titer, 7 IU at 10xe2x88x925 dilution and 10 percent TCGF, free of PHAxe2x80x9d). The reason for using the antiserum to xcex1-interferon was to neutralize endogenous interferon which is secreted by cells chronically infected by viruses, including retroviruses. In the mouse system, it had previously been shown that anti-serum to interferon could increase retrovirus production by a factor of 10 to 50 (F. Barre-Sinoussi et al., xe2x80x9cAnn. Microbiol. (Institut Pasteur)xe2x80x9d 130B, 349 (1979). After 3 days, the culture was continued in the same medium without PHA. Samples were regularly taken for reverse transcriptase assay and for examination in the electron microscope.
After 15 days of culture, a reverse transcriptase activity was detected in the culture supernatant by using the ionic conditions described for HTLV-I (B. J. Poiesz et al. xe2x80x9cProc. Natl. Acad. Sci. U.S.A.xe2x80x9d 77, 7415 (1980)). Virus production continued for 15 days and decreased thereafter, in parallel with the decline of lymphocyte proliferation. Peripheral blood lymphocytes cultured on the same way were consistently negative for reverse transcriptase activity, even after 6 weeks. Cytomegalovirus could be detected, upon prolonged co-cultivation with MRC5 cells, in the original biopsy tissue, but not in the cultured T lymphocytes at any time of the culture.
The invention relates to the newly isolated virus as a source of the above said antigen which will be defined later.
The newly isolated virus, which will hereafter be termed as LAV1, will however be described first.
The virus is transmissible to cultures of T lymphocytes obtained from healthy donors. Particularly virus transmission was attempted with the use of a culture of T lymphocytes established from an adult healthy donor of the Blood Transfusion Center at the Pasteur Institute. On day 3, half of the culture was co-cultivate with lymphocytes from the biopsy after centrifugation of the mixed cell suspensions. Reverse transcriptase activity could be detected in the supernatant on day 15 of the coculture but was not detectable on days 5 and 10. The reverse transcriptase had the same characteristics as that released by the patient""s cells and the amount released remained stable for 15 to 20 days. Cells of the uninfected culture of the donor lymphocytes did not release reverse transcriptase activity during this period or up to 6 weeks when the culture was discontinued.
The cell-free supernatant of the infected co-culture was used to infect 3-day-old cultures of T lymphocytes from two umbilical cords, LC1 and LC5, in the presence of Polybrene (2 xcexcg/ml). After a lag period of 7 days, a relatively high titer of reverse transcriptase activity was detected in the supernatant of both cord lymphocyte cultures. Identical cultures, which had not been infected, remained negative. These two successive infections clearly show that the virus could be propagated on normal lymphocytes from either new-borns or adults.
In the above co-cultures one used either the cells of patient 1 as such (they declined and no longer grew) or cells which had been pre-X-rayed or mitomycin C-treated.
The LAV1 virus, or LAV1 virus suspensions, which can be obtained from infected cultures of lymphocytes have characteristics which distinguish them completly from other HTLV. These characteristics will be referred to hereafter and, when appropriate in relation to the drawing. It shows curves representative of variation of reverse transcriptase activity and [3H] uridine activity respectively versus successive fractions of the LAV1 virus in the sucrose gradient, after ultra-centrifugation therein of the virus contents of a cell-free supernatant obtained from a culture of infected lymphocytes.
The analysis of LAV1 virus by resorting to reverse transcriptase activity can be carried out according to the procedure which was used in relation to virus from patient 1, on FIG. 1. The results of the analysis are illustrated on FIG. 1. Cord blood T lymphocytes infected with virus from patient 1 were labelled for 18 hours with [3H] urinde (28 Ci/mmole, Amersham; 20 xcexcCi/ml). Cell-free supernatant was ultra-centrifuged for 1 hour at 50,000 rev/min. The pellet was resuspended in 200 xcexcl of NTE buffer (10 mM tris, pH 7.4, 100 mM NaCl, and 1 mM EDTA) and was centrifuged over a 3-ml linear sucrose gradient (10 to 60 percent) at 55,000 rev/min for 90 minutes in an IEC type SB 498 rotor . Fractions (200 xcexcl) were collected, and 30 xcexcl samples of each fraction were assayed for, DNA fi,A dependant polymerase activity with 5 mM Mg2+ and poly(A)-oligo-(dT) as template primer; a 20-xcexcl portion of each fraction was precipitated with 10 percent trichloroacetic acid and then filtered on a 0.45-xcexcm Millipore filter. The 3H labelled acid precipitable material was measured in a Packard xcex2 counter.
That the new virus isolate was a retrovirus was further indicated by its density in the above sucrose gradient, which was 1.16, and by its labelling with [3H] uridine (FIG. 1). A fast sedimenting RNA appears to be associated with the LAV1 virus.
Virus-infected cells from the original biopsy as well as infected lymphocytes from the first and second viral passages were used to determine the optimal requirements for reverse transcriptase activity and the template specificity of the enzyme. The results were the same in all instances. The reverse transcriptase activity displayed a strong affinity for poly(adenylate-oligodeoxy-thymidylate) [poly(A)-oligo(dT)12-18], and required Mg2+ with an optimal concentration (5 mM) and an optimal pH of 7.8. The reaction was not inhibited by actinomycin D. This character, as well as the preferential specificity for riboseadenylate-deoxythymidylate over deoxyadenylate-deoxythymolylate, distinguish the viral enzyme from DNA-dependent polymerases.
Electron microscopy of ultrathin sections of virus-producing cells shows two types of particles, presumably corresponding to the immature and mature forms of the virus: immature particles are budding at the cell surface, with a dense crescent in close contact with the plasma membrane. Occasionally, some particles remain in this state, while being freed from the cell surface.
Mature particles have a quite different morphology with a small, dense, eccentric core (mean diameter: 41 nM). Most of virions are round (mean diameter: 139 nM) or ovoid, but in some pictures, especially in the particles seen in the original culture from which the virus was isolated, a tailed morphology can also be observed. The latter form can also be observed in cytoplasmic vesicles which were released in the medium. Such particles are also formed by budding from vesicle membranes.
Morphology of mature particles is clearly distinct from HTLV, whose large core has a mean diameter of 92 nM.
Helper T-lymphocytes (Leu 3 cells) form the main target of the virus. In other words the LAV1 virus has particular tropism for these cells. Leu 3 cells are recognizable by the monoclonal antibodies commercialized by ORTHO under the trademark OKT4. In contrast enriched cultures of Leu 2 cells, which are mainly suppressor or cytotoxic cells and which are recognized by the mono-clonal antibodies commercialized by ORTHO under the trademark OKT8 did not produce, when infected under the same conditions, any detectable RT activity even 6 weeks after virus infection.
In most cases of AIDS, the ratio of OKT4+ over OKT8+ cells which is normally over 1, is depressed to values as low of 0.1 or less.
The LAV1 virus is also immunologically distinct from previously known HTLV-1 isolates from cultured T lymphocytes of patients with T lymphomas and T leukemias. The antibodies used were specific for the p19 and p24 core proteins of HTLV-1. A monoclonal antibody to p19 (M. Robert-Guroff et al. xe2x80x9cJ. Exp. Med.xe2x80x9d154, 1957 (1981)) and a polyclonal goat antibody to p24 (V. S. Kalyanaraman et al. xe2x80x9cJ. Virol.xe2x80x9d, 38, 906 (1981)) were used in an indirect fluorescence assay against infected cells from the biopsy of patient 1 and lymphocytes obtained from a healthy donor and infected with the same virus. The LAV1 virus-producing cells did not react with either type of antibody, whereas two lines of cord lymphocytes chronically infected with HTVL 1  (M. Popovic, P. S. Sarin, M. Robert-Guroff, V. S. Kalyanaraman, D. Mann, J. Minowada, R. C. Gallo, xe2x80x9cSciencexe2x80x9d 219, 856 (1983))and used as controls showed strong surface fluorescence.
In order to determine which viral antigen was recognized by antibodies present in the patient""s sera, several immunoprecipitation experiments were carried out. Cord lymphocytes infected with virus from patient 1 and uninfected controls were labelled with [35S] methionine for 20 hours. Cells were lysed with detergents, and a cytoplasmic S10 extract was made. Labelled virus released in the supernatant was banded in a sucrose gradient. Both materials were immunoprecipitated by antiserum to HTVL-1 p24, by serum from patient 1, and by serum samples from healthy donors. Immunocomplexes were analyzed by polyacrylamide gel eletrophoresis under denaturing conditions. A p25 protein present in the virus-infected cells from patient 1 and in LC1 cells infected with this virus, was specifically recognized by serum from patient 1 but not by antiserum to HTLV-1 p24 obtained under similar conditions or serum of normal donors. Conversely the p24 present in control HTLV-infected cell extracts was recognized by antibodies to HTLV but not by serum from patient 1.
The main protein (p25) detected after purification of 35S-methionine-labelled virus has a molecular weight of about 25,000 (or 25KDa). This is the only protein recognized by the serum of patient 1. By analogy with other retroviruses, this major protein was considered to be located in the viral core.
This can be confirmed in immuno-electron microscopy experiments, which show that the patient""s serum can agglutinate the viral cores. Conversely, an antiserum raised in rabbit against an ether treated virus did not precipitate the p25 protein.
The viral origin of other proteins seen in polyacrylamide gel electrophoresisof purified virus is more difficult to assess. A p15 protein could be seen after silver staining, but was much weaker after 35S-methionine perhaps due to the paucity of this amino-acid in the protein. In the higher MW range, a contamination of the virus by cellular proteins, either inside or outside the viral envelope, is likely. A 36K and a 42K protein and a 80K protein were constantly formed to be associated with the purified virus and may represent the major envelope proteins.
No. p19 (or having a molecular weight of about 19 mM) was isolated from LAV1 extracts.
The invention concerns more particularly the extracts of said virus as soon as they can be recognized immunologically by sera of patients afflicted with LAS or AIDS. Needless say any type of immunological assay may be brought into play. By way of example immunofluorescence or immunoenzymatic assays or radio-immunoprecipitation tests are particularly suitable.
As a matter of fact and except under exceptional circumstances, sera of diseased patients do not recognize the intact LAV1 virus, or viruses having similar phenotypical or immunological properties. The envelope proteins of the virus appeared as not detectable immunologically by the patients"" sera. However as soon as the core proteins become exposed to said sera, the immunological detection becomes possible. Therefore the invention concerns all extracts of the virus, whether it be the crudest onesxe2x80x94particularly mere virus lysates xe2x80x94or the more purified ones, particularly extracts enriched in the p25 protein or even the purified p25 protein or in protein immunologically related therewith. Any purification procedure may be resorted to. By way of example only, one may use purification procedures such as disclosed by R. C. Montelaro et al, J. of Virology, June 1982, pp. 1029-1038.
The invention concerns more generally extracts of any virus having similar phenotype and immunologically related to that obtained from LAV1. Sources of viruses of the LAV type consist of T-lymphocyte cultures isolatable from LASxe2x80x94and AIDSxe2x80x94patients or, from haemophiliacs.
In that respect other preferred extracts are those obtained from two retroviruses obtained by propagation on normal lymphocytes of the retroviruses isolated from:
1) lymph node lymphocytes of a caucasian homosexual with multiple partners, having extensive Kaposi sarcoma lesions and severe lymphopenia with practically no OKT4+ lymphocytes in his blood;
2) blood lymphocytes of a young B haemophiliac presenting neurotoxoplasmosis and OKT4+/OKT8+ ratio of 0.1.
These two retroviruses have been named IDAV1 and IDAV2 respectively (for Immune Deficiency Associated Virus). Results of partial characterization obtained so far indicate similarityxe2x80x94if not identityxe2x80x94of IDAV1 and IDAV2 to LAV1:
same ionic requirements and template specificities of reverse transcriptase,
same morphology in ultrathin sections,
antigenically related p25 proteins: serum of LAV1 patient immunoprecipitates p25 from IDAV1 and IDAV2; conversely, serum from IDAV2 patient immunoprecipitates LAV1 p25.
IDAV1 patient serum seemed to have a lower antibodies titer and gave a weak precipitation band for LAV1 and IDAV1 p25 proteins. The p25 protein of IDAV1 and IDAV2 was not recognized by HTLV p24 antiserum.
These similarities suggest that all these three isolates belong to the same group of viruses.
The invention further relates to a method of in vitro diagnosis of LAS or AIDS, which comprises contacting a serum or other biological medium from a patient to be diagnosed with a virus extract as above defined and detecting the immunological reaction.
Preferred methods bring into play immunoenzymatic or immunofluorescent assays, particularly according to the ELISA technique. Assays may be either direct or indirect immunoenzymatic or immunofluorescent assays.
Thus the invention also relates to labelled virus extracts whatever the type of labelling: enzymatic, fluorescent, radioactive, etc.
Such assays include for instance:
depositing determined amounts of the extract according to the invention in the wells of titration microplate;
introducing in said wells increasing dilutions of the serum to be diagnosed;
incubating the microplate;
washing the microplate extensively;
introducing in the wells of the microplate labelled antibodies directed against blood immunoglobulines the labelling being by an enzyme selected among those which are capable of hydrolysing a substrate, whereby the latter then undergoes a modification of its absorption of radiations, at least in a determined wavelength band and
detecting, preferably in a comparative manner with respect to a control, the amount of substrate hydrolysis as a measure of the potential risks or effective presence of the disease.
The invention also relates to kits for the above said diagnosis which comprise:
an extract or more purified fraction of the abovesaid types of viruses, said extract or fraction being labelled, such as by a radioactive, enzymatic or immunofluorescent label;
human anti-immunoglobulins or protein A (advantageously fixed on a water-insoluble support such as agarose beads);
a lymphocyte extract obtained from a healty person;
buffers and, if appropriate, substrates for the vizualization of the label.